WO2020036341A1 - Combined use of squirrel poxvirus and myxoma poxvirus, for treating cancer - Google Patents

Abstract

The present invention relates to a composition and method for treating cancer in patients requiring a cancer treatment, and cancer can be treated by a combined therapeutic use of squirrel poxvirus and myxoma poxvirus.

Description

Combination of Squirrel Fox Virus and Myxomapox Virus for Cancer Treatment

The present invention relates to the therapeutic use of squirrelpox virus and myxomapox virus for the treatment of cancer.

This application claims priority based on US patent application Ser. No. 62 / 719,342, filed Aug. 17, 2018, and all content disclosed in the specification and drawings of that application is incorporated herein by reference.

Cancer is generally defined as disease, malignant tumors and neoplasms in which cells divide at an excessive rate and function abnormally. Standard therapies include surgery, chemotherapy and radiotherapy to remove affected cancerous tissue. Certain cancers do not respond or become resistant to such chemotherapy, radiotherapy, and other treatments. Although some human tumors show sensitivity to conventional chemo / radiation therapy, various solid tumors (such as brain tumors, breast tumors, ovarian tumors and other tumors) and blood tumors are known to be refractory to conventional treatment regimens. Leukemia is an exemplary cancer of the blood or bone marrow characterized by abnormal proliferation (production by proliferation) of blood cells, usually white blood cells (leukocytes). Chronic myeloid leukemia (CML) is a form of leukemia characterized by marked increase and unregulated growth of bone marrow cells in the bone marrow and accumulation of these cells in the blood. In addition, multiple myeloma (MM) is a highly refractory cancer and clonal B-cell neoplasm that affects the final differentiated B cells (ie plasma cells). In multiple myeloma, clusters of excess cancerous plasma cells can form tumors in the bone marrow, called myeloma. Therefore, the presence of many tumors is called multiple myeloma. The average survival rate for multiple myeloma patients is about 3 years. Genetic studies have shown that multiple myeloma tumorigenesis occurs through staged malignant transformation, including oncogene overactivation and inactivation of various tumor suppressor genes. Despite various drug treatments, cancer patients (CML or MM) often acquire rapid drug resistance and suffer from serious side effects caused by long-term drug treatment.

Naturally occurring viruses are viruses capable of living replication that specifically infect human cancer cells while avoiding their normal counterparts. Since the discovery of naturally antitumor viruses in the 1920s, a variety of cloned viruses have shown varying degrees of safety and efficacy in preclinical or clinical applications for anticancer therapies in humans or animals. Cell oncogenes such as Ras and c-Myc are known to be important host genes for determining oncolytic viral tropism. Importantly, cellular tumor suppressor genes have also been found to be important for the determination of oncolytic virus effects (Kim M et al., Oncogene. 29: 3990-3996, 2010). Carcinogenesis is a multistep process involving abnormal accumulation of oncogenes as well as abnormal accumulation of tumor suppressor genes. Therefore, it is interesting that oncolytic viruses are known to utilize abnormal oncogenes and cellular tumor suppressor signaling, often unregulated in gynecological malignancies, for their determination of oncogenic specificity and efficacy. Unlike oncogenes, many tumor suppressor genes such as p53, ATM, and RB are known to play an important role in genome fidelity / maintenance (Kim M et al., Oncogene. 29: 3990-3996, 2010). Tumor suppressor gene abnormalities may therefore affect host genome integrity and similarly may destroy intact antiviral networks due to the accumulation of gene defects that result in natural viral antitumor.

Since smallpox was eradicated in the 1970s, tumor-destructive properties of cloned vaccinia virus and other poxviruses have been identified. During the last 20 years of molecular research, the development of cellular oncogenes and tumor suppressor genes in determining poxvirus tumor destruction effects is now fairly well established. Currently, clinical trials are being carried out using modified versions of vaccinia virus strains in North America, Europe, and a few Asian countries. Although vaccinia virus complications could occur on a large population scale, including those in immunocompromised individuals, the safety aspects of attenuated versions of rare vaccinia viruses have now been fairly established through many experiments that have already been performed (Jacobs , BL et al, Antiviral Res. 84, 1-13, 2009; Nalca, A., Zumbrun, EE, Drug Des Devel Ther. 4: 71-79, 2010).

The present inventors confirmed that the combination of the squirrel fox virus and myxomapox virus exhibited a significant synergistic effect in the treatment of cancer, and based on this, the present invention was completed.

Accordingly, the problem to be solved by the present invention is a therapeutically effective amount of (a) a combination of squirrel fox virus and myxomapox virus; Or (b) providing a pharmaceutical composition for preventing or treating cancer, comprising the biological sample treated with (a) as an active ingredient.

In addition, another problem to be solved by the present invention is a therapeutically effective amount of (a) a combination of squirrel fox virus and myxomapox virus; Or (b) administering a biological sample treated with (a) to the patient, to provide a method of treating cancer in a patient in need thereof.

In addition, another problem to be solved by the present invention is a therapeutically effective amount of (a) a combination of squirrel fox virus and myxomapox virus; Or (b) providing a prophylactic or therapeutic use for cancer comprising the biological sample treated with (a) as an active ingredient.

In addition, another object of the present invention is to provide a kit comprising the pharmaceutical composition and an indication label for the composition.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned can be clearly understood by those skilled in the art from the following description. There will be.

In order to achieve the above object, in the present application (a) a combination of squirrel fox virus and myxomapox virus; Or (b) administering a therapeutically effective amount of a biological sample treated with (a) to the patient, the method of treating cancer in a patient in need thereof.

Also disclosed herein are therapeutically effective amounts of (a) a combination of squirrelpox virus and myxomapox virus; Or (b) a prophylactic or therapeutic use for cancer comprising the biological sample treated with (a) as an active ingredient.

The disclosure herein also relates to a biological sample treated with (a) a combination of squirrel fox virus and myxomapox virus, or (b) (a); And it provides a pharmaceutical composition for preventing or treating cancer comprising a pharmaceutically acceptable excipient. The pharmaceutical composition may include a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is an injectable dosage form. In some embodiments, the pharmaceutical composition is in oral dosage form. Also disclosed herein are indication labels for kits and compositions comprising the pharmaceutical compositions disclosed herein.

In some embodiments, the squirrel fox virus comprises a wild type squirrel fox virus. In some embodiments, the squirrel fox virus comprises a therapeutic gene-expressing squirrel fox virus for cancer targeting. In some embodiments, the myxomapox virus comprises a wild type myxomapox virus. In some embodiments, the myxomapox virus comprises a therapeutic gene-expressing myxomapox virus for cancer targeting. In some embodiments, the squirrel poxvirus may be contained 3x10 ⁵ to 10x10 ⁵ TCID _50. In some embodiments, the dynamic Soma poxvirus may be contained 0.1x10 ⁵ to 10x10 ⁵ TCID _50. In some embodiments, the squirrel fox virus and myxomapox virus titer ratio in the squirrelpox virus and myxomapox virus combination may be between 1: 1 and 100: 1 (squirrel foxx virus: myxomapox virus).

In some embodiments, the cancer may be cancer in which tumor suppressors are defective. In some embodiments, the cancer may be selected from hematopoietic malignancies, lung cancer, liver cancer, breast cancer, colon cancer, pancreatic cancer, brain cancer, gynecological cancer, or gastric cancer. In some embodiments, the cancer may be a hematologic malignancy selected from the group consisting of lymphomas, myeloma, myelodysplastic syndromes (MDS), myeloproliferative diseases such as hyperlipidemia, and leukemia. Cancer cells may be malignant cells or benign cells. In some embodiments, the cancer may be refractory cancer, including oncolytic virus resistant cancer.

In some embodiments, the biological sample is one prepared to kill multiple cancer cells by applying an effective amount of a squirrel fox virus and myxomapox virus combination to an ex vivo biological sample.

The combination of the squirrel fox virus and myxomapox virus according to the present invention exhibits a significant synergistic effect in the treatment of cancer.

1 shows the squirrel poxvirus oncolytic effects in gynecological carcinoma. EM magnification: 30,000x.

2 shows the squirrel poxvirus oncolytic effects in hepatocellular carcinoma. EM magnification: 60,000x.

3 shows the squirrel poxvirus oncolytic effects in gastric cancer. EM magnification: 30,000x.

4 shows the synergistic oncolytic effects of squirrelpox virus and myxomapox virus in Hep3B hepatocellular carcinoma. SQPV: Squirrel Fox virus; MYXVgfp: gfp expressing myxomapox virus.

5 shows the synergistic oncolytic effects of squirrelpox virus and myxomapox virus in A549 lung cancer. SQPV: Squirrel Fox virus; MYXVgfp: gfp expressing myxomapox virus.

6 shows the synergistic oncolytic effects of squirrelpox virus and myxomapox virus in MCF7 breast cancer cells. SQPV: Squirrel Fox virus; MYXVgfp: gfp expressing myxomapox virus.

7 shows the synergistic oncolytic effect of squirrel fox virus or myxomapox virus in squirrel fox virus resistant cancer. SQPV: wild type squirrel fox virus; MYXVgfp: gfp expressing myxomapox virus.

8A and 8B show synergistic tumor destruction effects of squirrelpox virus and myxomapox virus in an xenograft mouse model in vivo. vehicle: negative control; SQPV: Squirrel Fox virus; MYXVgfp: gfp expressing myxomapox virus.

Surprisingly, it has been found that the use of a combination of squirrelpox virus and myxomapox virus significantly improves the tumor destruction potential for human cancers, including tumor-resistant virus-resistant tumors. It can be shown to exhibit a significantly improved tumor destruction potential for human or animal cancer. Moreover, non-vaccinia poxviruses such as the squirrelpox virus and myxomapox virus are expected to be effective as they are nonpathogenic to humans and exhibit oncogenic potential for various cancers.

(A) a combination of squirrel fox virus and myxomapox virus; Or (b) administering a therapeutically effective amount of a biological sample treated with (a) to the patient, the method of treating cancer in a patient in need thereof.

Also disclosed herein are therapeutically effective amounts of (a) a combination of squirrelpox virus and myxomapox virus; Or (b) a prophylactic or therapeutic use for cancer comprising the biological sample treated with (a) as an active ingredient.

The disclosure herein also relates to a biological sample treated with (a) a combination of squirrel fox virus and myxomapox virus, or (b) (a); And it provides a pharmaceutical composition for preventing or treating cancer comprising a pharmaceutically acceptable excipient. The pharmaceutical composition may include a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is an injectable dosage form. In some embodiments, the pharmaceutical composition is in oral dosage form. Also disclosed herein are indication labels for kits and compositions comprising the pharmaceutical compositions disclosed herein.

The cancer may be cancer with tumor suppressor deficiency function. The cancer may be selected from hematopoietic malignancies, lung cancer, liver cancer, breast cancer, colon cancer, pancreatic cancer, brain cancer, gynecological cancer, or gastric cancer. The cancer may be a hematologic malignancy selected from the group consisting of lymphomas, myeloma, myelodysplastic syndromes (MDS), myeloproliferative diseases such as myelodysplastic disease, and leukemia. Cancer cells may be malignant cells or benign cells. The cancer may be refractory cancer, including oncolytic virus resistant cancer.

The term "squirrel fox virus" means a squirrel fox virus strain that is not pathogenic to human or non-squirrel animal species. Certain animal poxviruses affect only certain animal species (Kim M., 2015). In other words, the virus does not cause disease in humans or non-tropismic animals in nature. In some embodiments, the squirrel fox virus comprises at least one of a wild type squirrel fox virus strain or an engineered squirrel fox virus strain carrying a therapeutic gene for cancer targeting, such as an immune stimulating gene, an interferon related gene, an apoptosis related gene.

The term "myxomapox virus" means a myxomapoxvirus strain that is not pathogenic to human or non-rabbit animal species. Certain animal poxviruses affect only certain animal species (Kim M., 2015). In other words, the virus does not cause disease in humans or non-animal animals in nature. In some embodiments, the myxomapox virus comprises at least one of a wild type myxomapox virus strain or an engineered squirrel poxvirus strain carrying a therapeutic gene for cancer targeting such as an immune stimulating gene, an interferon related gene, an apoptosis related gene do.

Squirrelpox viruses and / or myxomapox viruses can be prepared using standard techniques known in the art (Kim M., 2015). Squirrelpox viruses and / or myxomapox viruses can be produced by processes that do not require high biosafety (BSL), and each virus is a unique progeny showing that viral genome replication occurs entirely in the cytoplasmic region of sensitive cells. Having a production cycle prevents the production of new hybrid viruses from the host human genome during cancer destruction by the combined virus therapy. As a result, manufacturing and drug development are feasible.

In some embodiments, the squirrel poxvirus may be contained 3x10 ⁵ to 10x10 ⁵ TCID _50. In some embodiments, the dynamic Soma poxvirus may be contained ⁵ to 10x10 ⁵ TCID 0.1x10 _50. In some embodiments, the squirrel fox virus and myxomapox virus titer ratio in the squirrelpox virus and myxomapox virus combination may be between 1: 1 and 100: 1 (squirrel foxx virus: myxomapox virus). More preferably, it may be 1: 1 to 10: 1 (squirrel foxx virus: myxomapox virus). Even more preferably, 5: 1 to 10: 1 (squirrel foxx virus: myxomapox virus).

Here, "TCID ₅₀ (Tissue culture infective dose ₅₀ )" is a virus quantification method also known as endpoint dilution assay. Typically, it is used to measure the virulence of eggs and animals of viruses that do not form plaques or specific viruses. In most cases, the virus dilution that infects 50% by inoculating 5 or more (usually 8-10 test units) cell monolayers, eggs, and animals in 10-fold dilutions of virus suspension is expressed as titer. Observe the cell monolayers inoculated with the virus at each fold to determine the presence of CPE and calculate the percentage of infected well. 50% endpoints are calculated using the Reed-Muench method or the Spearman-Karber method.

A "biological sample" is intended to mean any biological sample, such as adult stem cells (fat cells-derived stem cells, bone marrow stem cells) or umbilical cord blood stem cells, cell lines, tissue culture, or other cell sources obtained from an individual. Methods of obtaining tissue biopsies and body fluids from mammals are well known in the art. For example, adipocyte-derived stem cells can be pretreated with oncolytic viruses and they can be administered to cancer patients.

In some embodiments, a biological sample may be one prepared to kill a plurality of cancer cells by applying an effective amount of a squirrel fox virus and myxomapox virus combination to an ex vivo biological sample. In some embodiments, the biological sample is a bone marrow sample or a blood sample.

As used herein, the term "tumor disruption potential" includes cell death by lysis or apoptosis or other cell death mechanisms, in addition to making the cells unable to grow or divide.

As used herein, the term "subject" or "patient" means any individual member of the animal kingdom, including humans. Examples of mammals include, but are not limited to, any member of the mammalian class: humans, nonhuman primates such as chimpanzees, and other apes and monkey species; Farm animals such as cattle, horses, sheep, goats, pigs; Domestic animals such as rabbits, dogs, and cats. In some embodiments of the methods and compositions provided herein, the mammal is human or non-human.

As used herein, the term “treat” or “treating” means an approach for obtaining beneficial or desirable outcomes, including clinical outcomes. Beneficial or desirable clinical outcomes include, but are not limited to, measurable or not possible, alleviation or amelioration of one or more symptoms or conditions, reduction of disease extent, stabilization of disease state, prevention of disease development, disease spread Prevention, delayed or slowed disease progression, delayed or slowed disease initiation, amelioration or alleviation of disease state, and remission (partial or total). "Treating" may mean prolonging the survival of the patient than expected in the absence of treatment. In addition, “treating” may mean inhibiting the progression of a disease and temporarily slowing the progression of the disease, but this may involve permanently stopping the progression of the disease. As will be appreciated by those skilled in the art, the outcome may not be beneficial or undesirable if treatment results in greater adverse effects on the treated patient than any benefit affected by the treatment while ameliorating certain disease states. have. The present disclosure provides a method of treating cancer comprising administering to a patient in need thereof a therapeutically effective amount of a squirrelpox virus and myxomapox virus (“combination of two viruses”). The combination of the two viruses can be administered to the patient using standard methods of administration. In some embodiments, a combination of two viruses is administered to a disease site. Alternatively, a combination of the two viruses can be administered systemically. In some embodiments, the combination of two viruses is administered intratumorally or by the use of virus-carrier cells, such as by autologous tumor or cytokine-activated autologous normal hematopoietic cells. In various embodiments, the combination of two viruses can be administered orally or parenterally, or by any standard method known in the art. Parenteral administration includes intravenous, intraperitoneal, subcutaneous, intramuscular, epithelial, nasal, intrapulmonary, intrathecal, rectal and topical modes of administration. Parenteral administration may be by continuous infusion over a selected period of time.

When administered to a patient, an “effective amount” or “therapeutically effective amount” of a single virus or a combination of viruses is a cancer at a reasonable benefit / risk ratio that can be applied to any medical treatment or prevention at that dose and for a sufficient time. The amount necessary to alleviate, improve, mitigate, improve, stabilize, prevent proliferation, slow down or delay or heal progression. Effective dosage levels include the severity of the disease, the activity of the active agent, the age, weight, health and sex of the patient, the sensitivity to the active agent, the time of administration, the route of administration, and the rate of excretion of the composition of the present disclosure, the duration of treatment, the composition of the present disclosure and The drugs used simultaneously or in combination, the pharmacodynamic properties of the combination of viruses, the virulence and titer of the combination of the two viruses, and other factors known in the medical art can be measured. The pharmaceutical compositions of the present disclosure may be administered alone or in combination with other publicly known anticancer drugs or known ingredients such as those known to have anticancer activity. It is important to administer the composition in the smallest amount that can take full effect without causing side effects, taking into account all of the above factors.

In addition, one skilled in the art would be able to determine the appropriate amount of combination of two viruses (squirrel foxx virus and / or myxomapox virus) for administration based on these factors. The combination of the two viruses can be initially administered in a suitable amount that can be adjusted as needed, depending on the clinical response of the patient. The effective amount of the combination of two viruses can be determined according to the maximum amount of the combination of two viruses that can be administered experimentally and safely, and the minimum amount of the combination of two viruses producing the desired result.

Significantly higher doses of the combination of the two viruses may be required to produce the same clinical effect as would be achieved through injection at the disease site of the combination of the two viruses when administering the combination of the two viruses systemically. have. However, the appropriate dose level should be the minimum amount that will achieve the desired result. The concentration of the combination of two viruses to be administered will depend on the virulence of the particular strain (s) of the combination of two viruses to be administered and on the nature of the cell being targeted. In some embodiments, a dose of less than about 10 ⁹ plaque forming units (“pfus”) is administered to a human patient. In various embodiments, about 10 ² to about 10 ¹² pfu, about 10 ² to about 10 ⁹ pfu, about 10 ³ to about 10 ⁸ pfu, or about 10 ⁴ to about 10 ⁷ pfu may be administered in a single dose.

A therapeutically effective amount of a combination of viruses may be provided repeatedly depending on the effectiveness of the initial treatment regimen. Administration is typically given periodically while any response is monitored. It will be appreciated by those skilled in the art that doses lower or higher than the doses indicated above may be provided depending on the dosing schedule and the route chosen.

The combination of the two viruses may be administered alone or in combination with other therapies, including chemotherapy, radiation therapy, immunotherapy or other antiviral therapy. For example, a combination of the two viruses can be administered before or after removal due to surgery of the primary tumor or before, concurrently or after treatment such as administration of radiotherapy or conventional chemotherapeutic drugs.

To aid in administration, the combination of the two viruses can be formulated as a component of the pharmaceutical composition. It is appreciated that the compositions may routinely contain pharmaceutically acceptable concentrations of salts, buffers, preservatives and various matching excipients. For all delivery forms, a combination of the two viruses can be formulated in a physiological salt solution. The pharmaceutical composition may additionally contain other therapeutic agents, such as anticancer agents. In some embodiments, the composition comprises a chemotherapeutic agent.

The chemotherapeutic agent can be any agent that, for example, substantially exhibits an oncolytic effect on cancer cells or neoplastic cells of the patient and does not inhibit or reduce the killing effect of the combination of viruses. For example, chemotherapeutic agents include, but are not limited to, anthracyclines, alkylating agents, alkyl sulfonates, aziridine, ethyleneimine, metinelamine, nitrogen mustards, nitrosoureas, antibiotics, anti metabolites, folic acid analogs, purine analogs , Pyrimidine analogs, enzymes, podophyllotoxins, platinum-containing agents or cytokines. Chemotherapeutic agents may be those that are known to be effective against certain cell types that are cancerous or neoplastic. In some embodiments, chemotherapeutic agents such as thiotepa, cisplatin-based compounds, and cyclophosphamide are effective in the treatment of hematopoietic malignancies.

The proportion and identity of the pharmaceutically acceptable diluent is determined by the route of administration chosen, the match with the combination of viruses, and standard pharmaceutical practice. In general, pharmaceutical compositions will be formulated with ingredients that will not significantly impair the biological properties of the combination of the two viruses.

The pharmaceutical composition provides for the preparation of a pharmaceutically acceptable composition suitable for administration to a patient such that an effective amount of the active substance (squirrel foxvirus and / or myxomapox virus) is combined in a mixture with a pharmaceutically acceptable vehicle. It can be prepared by a known method for. Suitable vehicles are described, for example, in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA 1985). Based on this, the composition, although not entirely, is in combination with one or more pharmaceutically acceptable vehicles or diluents and of a combination of two viruses contained in a buffer solution having a suitable pH and having the same physiological fluid and osmotic pressure as one another. Solution.

The pharmaceutical composition can be administered to the patient in various forms depending on the route of administration chosen, as will be appreciated by those skilled in the art. The pharmaceutical compositions of the invention can be administered orally or parenterally. The pharmaceutical composition may be administered orally, for example, with an inert diluent or with an assimilation carrier, or may be enclosed in a hard or soft shell gelatin capsule, or compressed into tablets. For oral therapeutic administration, the combination of viruses can be integrated with excipients and used in the form of ingestible tablets, buccal tablets, troche tablets, capsules, elixirs, suspensions, syrups, wafers and the like.

Solutions of the combination of the two viruses can be prepared in physiologically suitable buffers. Under ordinary conditions of storage and use, these preparations contain a preservative that will prevent the growth of microorganisms but will not inactivate the combination of viruses. Those skilled in the art will know how to prepare suitable formulations. Conventional procedures and components for the selection and preparation of suitable formulations are described, for example, in Remington's Pharmaceutical Sciences and in The United States Pharmacopeia: The National Formulary (USP 24 NF19) (1999).

In some embodiments, the pharmaceutical composition is administered directly by injection (subcutaneously, intravenously, intramuscularly, etc.) to a disease site, such as a tumor site, or by oral administration, alternatively by transdermal administration. .

Forms of pharmaceutical compositions suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the instant preparation of sterile injectable solutions or dispersions, wherein the term sterile does not extend to the combination of viruses to be administered itself. Does not. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists.

The dosage of the pharmaceutical composition to be used depends on the specific disease being treated, the severity of the disease, the age, physical condition, size and weight of the individual patient parameter, the duration of treatment, the nature of the concurrent treatment (if any), the specific route of administration and the health personnel. It depends on other similar factors within the scope of knowledge and expertise. These factors are known to those skilled in the art and can be solved with minimal basic experimentation. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of the active agent (s) and a pharmaceutically acceptable excipient.

The combination of the two viruses of the invention is effective against cells from cancer, including oncolytic virus resistant cancer. As used herein, the term "cancer" or "cancer cell" refers to a cell that exhibits abnormal growth, characterized by a significant loss of control of cell proliferation or immortalized cells. The term "cancer" may include metastatic cancer, as well as non-metastatic cancer.

Exemplary cancers and / or cancer cells treatable by the present invention include, but are not limited to, hematopoietic malignancies such as lymphoma, myeloma, and leukemia, lung cancer, small cell lung cancer, breast cancer, colon cancer, pancreatic cancer, brain cancer, ovarian cancer and Cells derived from patients with gastric cancer. In some embodiments, the cancer cells treated by the combination of viruses described herein are from patients with hematologic malignancies. The hematologic malignancy can be leukemia, lymphoma, or myeloma. In some embodiments, the cancer cells are from a patient with one of CML and MM. In some embodiments, the cancer is refractory cancer and / or cancer cells are derived from “refractory cancer”. As used herein, “refractory cancer” is meant to denote a cancer that has not responded to or has become resistant to treatment.

Biological samples, including hematopoietic stem cells and cancer cells, may be obtained from a patient through any standard procedure known in the art, including but not limited to biopsies and aspiration. Once treated with the combination of viruses of the invention, the treated hematopoietic stem cells can be returned or administered to a patient using any known technique known in the art.

Previous studies have shown that oncolytic myxopox virus preferentially infects and kills tumor suppressor dysfunction cells (Kim M. et al., 2010). Not wishing to be bound by theory, the combination of viruses is normal because the combination of viruses is thought to kill cancerous cells that contain the p53, ATM and Rb tumor suppressor genes of dysfunctional cells and contain a defective interferon response. Cancer cells can be selectively killed without damaging human or animal tissue cells.

As used herein, “pharmaceutically acceptable” is relatively non-toxic, i.e., does not destroy the biological activity or properties of the active agent, i.e. any of the components of the composition in which the substance causes undesirable biological effects or contains it. Excipients, such as carriers or diluents, which can be administered to a subject without interacting in a deleterious manner are indicated.

Pharmaceutical compositions as used herein include other pharmaceutically acceptable chemical components, ie excipients such as, but not limited to, carriers, stabilizers, diluents, disintegrants, suspensions, thickeners, binders, antimicrobial agents, antimicrobial preservatives Combinations of viruses as active agent (s), including antioxidants, and / or buffers.

As used herein, the term “carrier” refers to a relatively nontoxic chemical compound or agent that facilitates incorporation of the active agent (s) into cells or tissues. The term "diluent" refers to a chemical compound used to dilute the active agent (s) of interest before delivery. Diluents can also be used to stabilize the active agent (s) because they can provide a more stable environment.

Once established, the buffer can be used to maintain the desired pH of the pharmaceutical composition from the effects of external agents and the equilibrium shift of the components of the composition.

In some embodiments, the pharmaceutical compositions of the invention are injectable dosage forms. For parenteral injection, suitable formulations include, for example, sterile aqueous or non-aqueous solutions containing physiologically compatible carriers.

In some embodiments, the pharmaceutical compositions of the invention are in oral dosage form. For oral administration, the active agent (s) described herein can be readily formulated by combining the active agent (s) with pharmaceutically acceptable carriers or excipients well known in the art. Such carriers enable the active agents described herein to be formulated as tablets, powders, pills, dragees, capsules, liquids, gels, syrups, elixirs, slurries, suspensions, and the like, for oral ingestion by a patient being treated.

The term “unit dosage form” refers to the entirety of a dosage regimen for producing a desired therapeutic effect, wherein each unit contains a predetermined amount of active ingredient in combination with a suitable pharmaceutical excipient, such as to treat cancer. As used throughout, physically distinct units suitable as single dosages are indicated.

The pharmaceutical compositions described herein may be in unit dosage forms suitable for single administration of precise dosages. In unit dosage form, the formulation is divided into unit doses containing an appropriate amount of one or more active agents. The unit dosage may be in the form of a package containing discrete amounts of the formulation. Non-limiting examples are tablets or capsules, and powders, packaged in vials or ampoules. The aqueous suspension composition can be packaged in a single-dose non-reseal container. Alternatively, a multi-dose reclosing container can be used, in which case it is typical to include a preservative in the composition. By way of example only, parenteral injection formulations may be provided in unit dosage form, including but not limited to, ampoules, or in multi-dose containers.

The aforementioned ranges are merely presented because of the large number of variables associated with the individual treatment regimen, and significant deviations from these recommended values are common. Such dosages depend on many variables, including but not limited to the activity of the active agent (s) used, the disease or condition being treated, the mode of administration, the requirements of the individual patient, the severity of the disease or condition being treated, and the judgment of the physician. can be changed.

The invention also relates to a sterile glass or polyolefin container comprising the pharmaceutical composition disclosed herein. In some embodiments, the container is a non-DEHP (bis (2-ethylhexyl) phthalate (di-2-ethylhexyl phthalate, diethylhexyl phthalate, DEHP; dioctyl phthalate, DOP) or non-PVP (polyvinylpyrroli Money).

The kit includes a suitable container such as a box, individual bottle, bag or ampoule. Suspension compositions may be packaged in single-dose non-resealable containers or multi-dose reclosable containers.

As used herein, the term "simultaneous administration" and the like is meant to encompass administration of two viruses (active agents) to a single patient, including therapeutic regimens in which the agents are administered by the same or different routes of administration or at the same time or at different times. It is intended to be.

Compositions containing the active agent (s) described herein can be administered for prophylactic and / or therapeutic treatments. In therapeutic applications, the composition is administered to a patient already suffering from a disease or condition in an amount sufficient to treat or at least partially arrest the condition or condition of the disease. The amount effective for such use will depend on the severity and course of the disease or condition, previous treatment, the patient's state of health, weight, and response to the active agent, and the judgment of the treating physician. Determination of such therapeutically effective amounts by basic experiments (including but not limited to dose escalation clinical trials) is believed to be within the skill of one of ordinary skill in the art.

In prophylactic applications, compositions containing the active agents described herein are administered to patients susceptible to or otherwise at risk of cancer. Such amount is defined as "prophylactically effective amount or dose." In such use, the exact amount also depends on the patient's state of health, weight, and the like. It is believed that determining such prophylactically effective amounts by basic experiments (such as dose escalation clinical trials) is within the skill of one of ordinary skill in the art. When used in a patient, an effective amount for such use will depend on the severity and course of the disease, disorder or disease, previous therapy, the patient's health condition and response to the active agent, and the judgment of the treating physician.

If the condition of the patient does not improve, at the physician's discretion, the administration of the active agents described herein may be chronically ie to improve or otherwise control or limit the patient's disease or symptoms of the disease. It may be administered for an extended period of time, including the entire life span.

The amount of a given agent that will correspond to that amount will depend on factors such as the particular active agent, the disease state and its severity, the identity of the subject or host in need of treatment (eg, age, weight, gender, etc.), but nevertheless And the specific circumstances surrounding the case, including, for example, the particular active agent administered, the route of administration, the disease being treated, and the subject or host being treated.

Example

Example 1: Squirrel poxvirus tumor destruction effect in gynecological carcinoma.

As shown in FIG. 1, wild-type squirrel fox virus was detected in the cytoplasmic region of C33A human cervical cancer cells upon squirrel fox virus challenge 2 to 4 days after infection (10 MOI). Mature / mature squirrelpox virus particles appear on high power electron microscopy (EM). EM magnification: 30,000x.

Example 2: Squirrel poxvirus tumor destruction effect in hepatocellular carcinoma.

As shown in FIG. 2, wild-type squirrel fox virus is detected in the cytoplasmic region of Hep3B human cervical cancer cells upon challenge with squirrel fox virus 2 to 4 days after infection (10 MOI). Mature / mature squirrelpoxvirus particles appear on high power electron microscopy (EM). EM magnification: 60,000x.

Example 3: Squirrel poxvirus tumor destruction effect in gastric cancer.

As shown in FIG. 3, wild-type squirrel fox virus was detected in the cytoplasmic region of AGS3 human gastric cancer cells upon squirrel fox virus challenge 2 to 4 days after infection (10 MOI). Mature / mature squirrelpoxvirus particles appear on high power electron microscopy (EM). EM magnification: 30,000x.

Example  4: in hepatocellular carcinoma Squirrel Fox  Virus and MIXOMAPOX  Synergistic oncolytic effect of virus.

Hep3B cells were infected with wild-type squirrel poxvirus (8x10 ⁵ TCID ⁵⁰⁾ or Mick Soma poxviruses (1x10 ⁵ TCID ^50). Six days after virus infection, cell colonies were fixed and stained with crystalline violet solution. As shown in FIG. 4, colonies were photographed using a high power scanner. Hep3B hepatocytes were killed by SQPV virus infection and MYXV-gfp virus infection. As the number of dots in the picture decreases, more cancer cells are killed. It was confirmed that the number of dots was significantly reduced by the combination of SQPV virus and MYXV-gfp virus. SQPV: Squirrel Fox virus; MYXVgfp: gfp expressing myxomapox virus.

Example  5: from lung cancer Squirrel Fox  Virus and MIXOMAPOX  Synergistic oncolytic effect of virus.

A549 cells were infected with wild-type squirrel poxvirus (8x10 ⁵ TCID ⁵⁰⁾ or Mick Soma poxviruses (1x10 ⁵ TCID ^50). Six days after virus infection, cell colonies were fixed and stained with crystalline violet solution. As shown in FIG. 5, colonies were taken using a high power scanner. A549 lung cells were killed by SQPV virus infection and MYXV-gfp virus infection. As the number of dots in the picture decreases, more cancer cells are killed. It was confirmed that the number of dots was significantly reduced by the combination of SQPV virus and MYXV-gfp virus. SQPV: Squirrel Fox virus; MYXVgfp: gfp expressing myxomapox virus.

Example  6: in breast cancer cells Squirrel Fox  Virus and MIXOMAPOX  Synergistic oncolytic effect of virus.

Infecting MCF7 cells with either wild-type squirrelpox virus (8x10 ⁵ TCID ⁵⁰ ) or myxomapox virus (1x10 ⁵ TCID ⁵⁰ ) or a combination of both viruses (SQPV: 4x10 ⁵ TCID ⁵⁰ , MYXVgfp: 0.5x10 ⁵ TCID ⁵⁰ ) I was. Six days after virus infection, cell viability was assessed using the WST-1 assay method as described in the manufacturer, as shown in FIG. 6. MCF7 breast cancer cells were killed with SQPV virus infection and MYXV-gfp virus infection. As a result, as compared with the case of treating a single virus, as a result of combining two viruses it was confirmed that there is a synergistic tumor destruction effect. SQPV: squirrel fox virus, MYXVgfp: gfp expressing myxomapox virus.

MCF7 MTT assay
				Ave	SD
Mock	1.6	1.4	1.8	1.6	0.2
SQPV	0.4	0.39	0.41	0.4	0.01
MYXVgfp	0.59	0.58	0.6	0.59	0.01
SQPV + MYVXgfp	0.3	0.29	0.31	0.3	0.01

Example  7: Squirrel Fox  In virus-resistant cancer Squirrel Fox  Virus or MIXOMAPOX  Virus synergistic oncolytic effects.

Squirrelpox virus resistant HTR1 cells (Kim et al., 2007) were either wild type squirrelpox virus (8x10 ⁵ TCID ⁵⁰ ) or myxomapox virus (1x10 ⁵ TCID ⁵⁰ ) or a combination of two viruses (SQPV: 4x10 ⁵ TCID). ⁵⁰ , MYXV or MYXVgfp: 0.5 × 10 ⁵ TCID ⁵⁰ ). Six days after virus infection, cell colonies were fixed and stained with crystalline violet solution. As shown in FIG. 7, colonies were taken using a high power scanner. As the number of dots in the picture decreases, more cancer cells are killed. The combination of SQPV and MYXV-gfp virus infection synergistically increased the death of HTR1 cancer cells. SQPV: wild type squirrel fox virus, MYXVgfp: gfp expressing myxomapox virus.

Example  8 : In vivo B16F10  In the rising model Squirrel Fox  Virus and MIXOMAPOX  Synergistic oncolytic effect of virus.

Age-matched (5 weeks postnatal) female C57BL / 6 mice were inoculated subcutaneously into the right flank with B16F10 murine melanoma cells (1 × 10 ⁵ cells per mouse). Mice were randomly assigned to treatment groups when the mean of tumor volumes reached approximately 100-120 mm ³ . Virus (squirrel foxx virus and myxomapox virus) or PBS vehicle was administered intratumorally on days 1, 2, 8 and 9. Tumor volume (FIG. 8A) and mouse body weight (FIG. 8B) were measured at regular intervals until tumors reached approximately 2,000 mm ³ . Combination treatment of squirrelpox virus and myxomapox virus was performed on B16F10 melanoma tumors, squirrelpox virus single infection (filled triangle, third line from the top) and myxomapox virus single infection (filled rectangle, second line from the top) Tumor growth inhibition was synergistically induced (lowest line) in comparison with. No signs and symptoms of systemic toxicity were observed in mice treated with two viruses that combined squirrelpox virus and myxomapox virus, compared to those in vehicle-treated mice as shown in FIG. 8B, Either single treatment or combination treatment was found to be non-toxic to virus infected mice. SQPV: Squirrel fox virus, MYXV gfp: gfp expressing myxomapox virus.

The teachings of the references cited throughout the specification are hereby incorporated by reference in their entirety to the extent that they do not contradict the teachings herein. It is to be appreciated that the examples and embodiments described herein are for illustrative purposes only and in that respect various modifications or changes will be presented to those skilled in the art and are intended to be included within the spirit and scope of the present application.

The foregoing description of specific embodiments can be adapted to various applications and / or easily modified such specific embodiments by applying knowledge within the skill of the art, without departing from the general inventive concept of others without unnecessary experimentation. It will reveal the general nature of the invention which can be adjusted for. Therefore, such adjustments and variations are intended to be within the meaning and scope of equivalents of the disclosed embodiments, based on the teachings and guidance provided herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, that the terminology or phraseology herein may be interpreted by one of ordinary skill in the art in view of teaching and guidance.

The breadth and scope of the present disclosure should not be limited by any of the above described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.

All of the various aspects, embodiments, and options disclosed herein can be combined in any and all variations.

All publications, patents, and patent applications mentioned herein are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

Claims (19)

1. Therapeutically effective amount of (a) a combination of squirrelpox virus and myxomapox virus; Or (b) a pharmaceutical composition for preventing or treating cancer, comprising the biological sample treated with (a) as an active ingredient.
2. The method of claim 1,

   The squirrel fox virus is a wild type squirrel fox virus, characterized in that the pharmaceutical composition.
3. The method of claim 1,

   Wherein said squirrel fox virus is a therapeutic gene-expressing squirrel fox virus for cancer targeting, pharmaceutical composition.
4. The method of claim 1,

   The myxomapox virus is a wild type myxomapox virus, characterized in that the pharmaceutical composition.
5. The method of claim 1,

   Wherein said myxomapox virus is a therapeutic gene-expressing myxomapox virus for cancer targeting.
6. The method of claim 1,

   The squirrel fox virus is characterized in that it comprises 3x10 ⁵ to 10x10 ⁵ TCID ₅₀ , pharmaceutical composition.
7. The method of claim 1,

   The myxomapox virus is characterized in that it comprises 0.1x10 ⁵ to 10x10 ⁵ TCID ₅₀ , pharmaceutical composition.
8. The method of claim 1,

   Pharmaceutical composition, characterized in that the squirrel fox virus and myxomapox virus titer ratio in the combination of squirrel fox virus and myxomapox virus is 1: 1 to 100: 1 (squirrel fox virus: myxomapox virus) .
9. The method of claim 1,

   Wherein said biological sample is prepared to kill a plurality of cancer cells by applying an effective amount of a combination of squirrel fox virus and myxomapox virus to an ex vivo biological sample.
10. The method according to claim 1 or 9,

    Wherein said biological sample is a bone marrow sample or a blood sample.
11. The method according to any one of claims 1 to 9,

    The cancer, characterized in that the cancer has a tumor suppressor deficiency function, pharmaceutical composition.
12. The method according to any one of claims 1 to 9,

    The cancer is characterized in that selected from hematopoietic malignancy, lung cancer, liver cancer, breast cancer, colon cancer, pancreatic cancer, brain cancer, gynecological cancer, or stomach cancer.
13. The method according to any one of claims 1 to 9,

    Wherein said cancer is a hematologic malignancy selected from the group consisting of lymphoma, myeloma, myelodysplastic syndrome (MDS), myelocytosis, and leukemia.
14. The method according to any one of claims 1 to 9,

    Wherein said cancer comprises malignant cells and / or benign cells.
15. The method according to any one of claims 1 to 9,

    The cancer is characterized in that the refractory cancer, including oncolytic virus resistant cancer, pharmaceutical composition.
16. The method according to any one of claims 1 to 9,

    The pharmaceutical composition, characterized in that the injectable dosage form.
17. The method according to any one of claims 1 to 9,

    The pharmaceutical composition is characterized in that the oral dosage form, pharmaceutical composition.
18. Therapeutically effective amount of (a) a combination of squirrelpox virus and myxomapox virus; Or (b) a prophylactic or therapeutic cancer comprising a biological sample treated with (a) as an active ingredient.
19. 10. A kit comprising the pharmaceutical composition according to any one of claims 1 to 9 and an indication label for the pharmaceutical composition.

Images